1. Field of the Invention
The present invention relates to a high performance liquid chromatography for separating and analyzing the various compounds in a sample, and an analytical method of the same, and a flow path switching valve used by the same.
2. Description of Related Art
As for mass spectrometers used for determining the structures of proteins or peptides in the field of life science, in order to optimize the sensitivity, it is required to minimize the flow in the high performance liquid chromatograph (HPLC) served as the previous stage of a mass spectrometer.
Generally, the HPLC employed in an ordinary analysis is operated by feeding a liquid as a mobile phase at a flow rate of about 1 mL/min into a column with an inner diameter of about 4.6 mm. However, as for mass spectrometers, Micro-HPLCs for feeding a liquid at a flow rate of about 5 μL/min into a column with an inner diameter of about 0.3 mm have been gradually applied. In addition, Nano-HPLCs for feeding a liquid as the mobile phase at a flow rate of about 200 nL/min into a column with an inner diameter of 0.075 mm have been gradually commercialized.
When the extremely low-flow HPLCs are used for analysis, the sample may be diffused due to the volume (about 100 μL) in the system, thus affecting the sensitivity of the mass spectrometer. Therefore, after the sample is injected into the system with an auto-injector or a manual injector, the sample is adsorbed in a trap column, connected to the flow path switching valve, for being condensed. Then, the flow path switching valve is switched to feed the mobile phase liquid used for analysis into the trap column, so that the sample adsorbed on the trap column is removed from the trap column, and then separated through an analytical column in a latter stage. Finally, the mass of the sample is analyzed by a mass spectrometer.
The flow path switching valve employed in an extremely low-flow HPLC has a small inner volume due to the same reason as that mentioned above. Generally, the main components of a flow path switching valve include a housing cover for fixing the pipes of the flow path switching valve, a rotor for switching flow paths through rotation, and a stator, disposed between the housing cover and the rotor, for keeping liquid-tightness. In the extremely low-flow HPLC, the flow path switching valve with a stator and a housing cover being integrated as a whole is generally used, so as to reduce the inner volume of the valve.
FIGS. 3A-3B are a flow path diagram of an HPLC using a flow path switching valve in the conventional art, in which FIG. 3A shows the flow path when the sample is condensed; and FIG. 3B shows the flow path when the sample is analyzed.
Each port of the flow path switching valve 21 is respectively connected through the flow paths to a trap column 5 for condensing the sample, a sample injection portion 9 for injecting the sample into the flow path between a pump 7 and the trap column 5, an analysis infusion pump 11 for feeding the mobile phase liquid used for the analysis in order to separate the ingredients already condensed by the trap column 5, and an analytical column 13 for separating the ingredients already condensed by the trap column 5. The analytical column 13 includes a detector 14 connected thereto in the downstream.
On the rotor of the flow path switching valve 21, three circular rotor slots 21a-21c are formed, as shown in FIGS. 3A and 3B. By means of rotating the rotor for 60 degrees, the combination of the rotor slots and the corresponding ports can be switched, so that the sample condensation and the sample separation and analysis can be performed.